AU661762B2 - Method for producing carbohydrates - Google Patents

Abstract

The invention relates to oligosaccharides with the sequence GalNAc beta (1-4) (Fuc alpha (1-3) GlcNAc. They are produced by incubating Human Kidney 293 cells and isolating the novel oligosaccharides from the peptide produced. oligosaccharides can be used to act as high affinity ligands of certain cell adhesion receptors, and may be used to prevent or reduce cell adhesion and inflammation.

Description

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XUSTRALIA

I TS A;T 1990 FQ ASTANA'$ PATENT AR FA.

I L- T- Name and Address of Applica.nt: Actual Inventor(s): Address for Service: Invention Title: Eli Lilly and Company Lilly Corporate Cebter City of Ind:ianpolis State of I.ndiana UNITED STATES OF AMERICA Sau-Chi Betty Yan Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Method For Producing Carbohydrates 4I C 14 9* 9 4 .9 9 0 U 1 e The following statement is a full description of this invention, Including the best method of performing it known to me/us:- 999 09 5845/3

F-.

tilt 1t '3 00 0 1y[0 a 0 2 C0 0 0 00 0c 00 00 0 0 0 C,0 0 Methud For Producing Cabhydrates 00 'The invention relates to molecular biology, particularly to methods for thie production of novel carbohydrate t uctures in human kidney 293 cells. These novel-, :carbohydrate structures can be uised to treat inflammation and can also be used to reduce cell adhesion.

Human Kidney 293 cells are 'denoviru-s-transformed cells useful in producing recombinant )proteins. The cells, have been found to be particularly useful for producing .blood pr~tein, products such~ a's hufan protein C, human protein S and thrombomodulin, as well as tissue p~lasminogen activatar, various derivative forms of human protein C and jo renin. Humran protein C proiced in 293 cells demongtrates a glycosylation pattern Whidh is markedly distinct from the glycosylation pattern found on plasma-derived human protein C. This invention relates to the fact that the human protein C molecules produced in 293 cells have now been Shown to contain novel N-linked aligosaccharides which contain mon7reducing terminal branches of .GalNAcp(1-4) ;(Fuca(1-3))GldNAc. These stijttas are capable of binding with high affinity to certain selectins, which are cell :adhesion receptors of the human immune system. This specific ligand affinity demonstrates that the novel oligosaccharides produced by 293 cells are -useful in the reduction of cell adhesion and tissue inflammation.

According to a first embodiment of this invention, there is provided an oligosacchaide of the structure Fuc~x(1-*3) GalNAcp(l *4)GcNAcj3(l -*2)Manx(l -6 fcLl->6) Manl3(l ->4)GlcNAcP(1-*4)GIcNAcI GaNAcP(1 ->4)GlcNAcP (1->2)Manc(l+ According to a second embodiment of this invention, there is prOvided an oligosaccharide of the structure Fuca(l 3) GaNAcP(l -*4)GlcNAc3(i -*2)Manac(l Fufcc(l Manf3(l ->4)GcNAc3(1-*4)GlcNAc According to a third embodiment of this invention, there is provided an oligosaccharide of the structure [N:\LIBVVIOO439:KEH

I-

0 K) c'-p F-)0 o 0 0 o 0 0~

CC

(0 C) 0 NeuAcat(2-* 6)GyaNP~cW1-*4)GcNAc3(17. 7*c)ManL(1 Fuca~( -46) Mar( ~iGcN.Ac3(1 -*4)GIcNAc I 0 GalNAc3(1 -*4)GcNAc3(1 -*2)Mamxal

CD

00 (-3 0

C

(C

According to a fourth embodiment of this invention, ethere is provided an oii $acharide of the struicture Nei~cct(2-~)Ga[3( 4GlcN~cf(1 ~2)ana( Tcc'q iMan3(1 -*4)GjcNAcf3(1-*4)GIcNAc Ga4NAP(-4)GINAcP1-4 2Yanc('i-+3) Fuca.(1 According to a fifth embodiment of this invention, ther is oided a pharmnaceutical formulation compris-ing as an active ingredient an oligosaccharide of the first, :second, third or fourth embodiment, associated with one or -more pharmaceutically acceqptable cearriers, excipients or diluents thejrefor.

According to a sixth embodiment of this inventiona, -there is provided a method of 1.0 treating inflamnmation in a mammal Which comtprises administering to that mammal an oligosacharide of the first, second, third or fourth embodiment.

According to a seventh embodiment of this invention, there is provided a method of treating cellular adhesion in a mammal which comprises administering to that mammal an olJigosaccharide of the first, second, third or fourth embodiment.

For purposes of the present invention, as disclosed and claimed herein, the following terms are as defined below.

Asn asparagine.

Gal galactose.

Fuc fucose.

GalNAc -N-acetylgalactosai-ine.

4o [N:\LIBVV]00439: 0

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Q0 0 Q0c0o 0 c0 o o 0 0 o 0 X-8833 GlcNAc N-acetylglucosamine.

Man Mannose.

NeuAc N-acetylneuraminic acid.

Figure 1. shows a chromatograph of the major carbohydrates found on the human protein C molecules produced in 293 cells. The major peaks for which carbohydrate structures have been elucidated are marked with numbers.

The present invention comprises a method for producing peptides containing oligosaccharides with the structure GalNac3(l-4) (Fuca(1-3) )GlcNAc by incubating Human Kidney 293 cells expressing a peptide capable of being glycosylated under conditions suitable for expression of the peptide, then recovering the peptide from the cell or cell culture supernatent. Human Protein C molecules produced in 293 cells contain these structures on the nonreducing terminal branches of N-linked oligosaccharides.

The invention also comprises methods for further isolating the novel oligosaccharides from the peptide produced in 293 cells. The peptides and/or oligosaccharides so produced can be used to act as high affinity ligands of certain cell adhesion receptors. Such ligand specificity demonstrates that the peptides and novel oligosaccharides may be used to prevent or reduce cell adhesion and thereby prevent or reduce tissue inflammation.

Human Kidney 293 cells (ATCC CRL 1573) are transformed primary embryonal human kidney cells which contain and express the transforming gene of Adenovirus 30 These cells have been used to express several important gene products and have been used by a number of different laboratories both in academia and industry. For example, IYan, U.S. Patent No. 4,981,952 and Bang et. al., U.S.

Patent No. 4,992,373, both disclose the use of the 293 cell line to produce human protein C. It has now been .r _L.

o o (Tl- O o oo o o- o 0 0 O0 Oo 0 o o o O o O 0 o 01o X-8833 -3- 0 discovered that the 293 cell line contains the enzymatic o 0 0 machinery to create novel and useful oli:gosaccharides on peptides produced in the cell line. These novel oligosaccharides, as well as methods for making and using them, comprise the present invention.

Human Protein C (IPC) circulates in th- plasma as a zymogen of a serine protease. Protein C is activated by thrombin in complex with the endothelial cell surface receptor, thrombomodulin. The activated form of human protein C (aPC) has potent anticoagulant activity due to its ability to inactivate factors Va and Villa. For purposes of the present disclosure, the term "human protein C" refers to both the zymogen and activated forms of the molecule. Protein C plays a critical role in the regulation of thrombin generation and may be effective in the treatment of a number of thrombotic diseases. Human Protein C is post-translationally modified by Nf"i, glycosylation at four sites on the molecule; specifically at position Asn 97 on the light chain, and at positions Asn 1,120 248, Asn 313, and Asn 329 on the heavy chain. While the full carbohydrate structures of plasma-derived HPC have not been reported, the oligosaccharides found on HPC recombinantly-produced in 293 cells (rHPC) are quite distinct from those found on plasma-derived HPC. For example, rHPC has a five-fold higher fucose content and a two-fold lower NeuAc content in comparison to plasmaderived HPC. In addition, there are 2.6 moles of GalNAc per mole of rHPC while plasma-derived HPC contains no GalNAc.

:30 The present invention is not limited to specific oligosaccharides linked to a peptide at an Asn residue.

Oligosaccharides may be linked to peptides via Nglycosylation or 0-glycosylation. Oligosaccharides are also found within glycolipids and proteoglycans and may be added to membrane bound proteins via glypiation. For the L I i Ir Ir I o 0 o C C 0 0 X-8833 e o -4r purposes of Ehis disclosure, the term "capable of being glycosylated" is not limited to N-glycosylation of an Asn residue, but rather includes all mechanisms by which peptides or lipids are glycosylated in vivo.

The major oligosaccharides found on rHPC produced in 293 cells display the profile shown in Figure 1 upon fingerprinting on the Dionex HPAE-PAD system. The oligosaccharide corresponding to major peak 1 is a neutral, biantennary oligosaccharide with both antennae containing the structure of GalNAc(l-4)(Fuca(l-3))GlcNAc(l-2)linked to the fucosylated chitobiose-trimannosyl core.

Several of the other ten oligosaccharides studied and disclosed herein also display the novel N-linked structure containing GalNAc. The predicted structures of the oligosaccharides of peaks 2, 7 and 11 also contain the novel structure on at least one branch of the fucosylated chitobiose-trimannosyl core. In addition, the structures of the oligosaccharides of peaks 15, 20 and 23 contain GalNAc residues in 1-4 linkage. These novel oligo- 20 saccharide structures are produced by the 293 cell line because the cells contain the specific enzymes necessary for production of these substrates.

Human Kidney 293 cells appear to have both a(2- 3) and a(2-6) sialyltransferases. The a (2-6) 25 sialyltransferase is specific for sialylating GalNAc residues while the a(2-3) sialyltransferase is specific for sialylating Gal residues. The a(l-3) fucosyltransferase expressed by 293 cells can act on a substrate containing GalNAc. The action of the a(2-6) sialyl- 0.0 .30 transferase and the a(i-3) fucosyltransferase appear to be mutually exclusive on any individual branch of the oligosaccharide, therefore once the GalNAc is sialylated the a fucosylation of the GalNAc residue in the same I antenna is precluded. Conversely, once the GalNAc is o o 0 Q u O o 0 X-8833 fucosylated, the same GalNAc residue on the same antenna cannot be sialylated by the a(2-6) sialyltransferase.

The 293 cells produce recombinant glycoproteins with less heterogeneity in the carbohydrate portion of the molecule because 293 cells express very high levels of a fucosyltransferase activity. The linkage analysis of all 25 oligosaccharide peaks isolated from rHPC revealed only 4,6-linked GlcNAc with a reducing end. No 4-linked GlcNAc with a reducing end was detected. From this it is o 10 apparent that the chitobiose cores of all the N-linked oligosaccharides were virtually 100% fucosylated. The elucidation of the 11 major oligosaccharides on 293 cellderived rHPC accounts for the unusual glycosyl content of the rHPC as reported by Yan et al., 1990, BioTechnoloov 8:655-660. The high fucose content of the molecule can be accounted for by the fact that the chitobiose core of all of the N-linked oligosaccharides are fucosylated and also by the fact that the GlcNAc residues in the antenna(e) of four of the 11 major N-linked oligosaccharides are fucosylated. With the exception of the oligosaccharide found in peak 2, the GalNAc residues in rHPC were all found in N-glycosylated oligosaccharides and were located only in the antennae position where normally only Gal is found in o N-linked complex type oligosaccharides. For purposes of 25 this disclosure, the terms antenna and branch can include the plural antennae and branches and vice versa.

0. The action of the fucosyltransferase found in 293 cells and the resulting a(l-3)fucosylation of the As GlcNAc residues of oligosaccharides made in the cell line demonstrate yet another facet of the present invention.

Structures containing a(l-3)fucosylated GlcNAc are useful ligands for the binding of selectins. Selectins are a type of adhesion receptor of the immune system. The adhesion of leukocytes to the endothelial lining is an integral step in 0 0 0 0 a 0 0 0 o 00° 0 0 o 0 0 0 X-8 33 o o the inflammatory response. Two types of selectins, CD62 and ELAM-1 (Endothelial-Leukocyte Adhesion Molecule 1) are bound by oligosaccharides containing (1-3)fudosylated GlcNAc. Cell adhesion assays demonstrate that the addition of peptides containing such novel oligosaccharides (or the addition of the oligosaccharides alone) can bind the selectins and therefore reduce or prevent cellular adhesion. If cellular adhesion is reduced or prevented, 0 the inflammatory response can also be reduced or prevented.

The discovery that 293 cells contain the enzymatic machinery necessary to produce such selectinlacognition ligands adds an important and useful tool to the medical community for the treatment of disease states linked to cell adhesion and the inflammatory response.

Because of this discovery, glycoproteins and oligosaccharides produced in 293 cells may be used to treat inflammation or cell adhesion. Methods for using rHPC to Streat a variety of health disorders are disclosed in Bang et al., U.S. Patent No. 4,775,624. The i vel glycoproteins S.20 produced by 293 cells are not limited to recombinantly produced proteins in that some glycoproteins produced constitutively by 293 cells may also display the novel structures. Furthermore, the invention is not limited to *the use of glycopeptides to prevent or reduce cell adhesion and inflammation. The skilled artisan will realize that the oligosaccharides can be removed from the glycoproteins by a wide variety of well-known procedures. The o, o oligosaccharides can then be placed into a pharmaceutically Sacceptable diluant and administersd Lo a patient undergoing cell adhesion or inflammation.

The skilled artisan will understand that the present invention is not limited to the production of the oligosaccharides of human protein C in 293 cells. Any peptide capable of being glycosylated can be transformed into 293 cells and expressed using techniques well known in -i I SOo 0 0 0 0 0 0 o a o 0 o o 0 0 So X-C8833 0 0 0 the art. A list of such peptides includes, but is not oo o limited to, human protein S, human thrombomodulin, o 0 derivatives of human protein C, tissue plasminogen o c activator and renin. The production of human protein S in 0 293 cells was disclosed in Yan, U.S. Patent No. c 4 98 1 952 o The genes encoding several derivatives of human protein C o are disclosed in European Patent Application No. o0~i 91301450.2. The genes encoding natural and derivative forms of human thrombomodulin have been disclosed in .European Patent Application No. 90308826.8 and Wen et al.,1987, Biochemistry 26:4350. Any of these gefde may be transformed into 293 cells which can then be cultured under conditions suitable for gene expression and peptide/oligosaccharide production.

The following examples are provided as a means of illustrating the present invention and are not to be 7 construed as a limitation thereon.

Example 1 0 .o Production of Human Protein C in 293 Cells J ".c 0 O Recombinant human protein C (rHPC) was produced in Human Kidney 293 cells by techniques well known to the 0.

skilled artisan such as those set forth in Yan, U.S. Patent o No. 4,981,952. The gene encoding human protein C is o disclosed and claimed in Bang et al., U.S. Patent No.

4,775,624. The plasmid used to express human protein C in 293 cells was plasmid pLPC which is disclosed in Bang et al., U.S. Patent No. 4,992,373. The construction of plasmid pLPC is also described in European Patent Publication No. 0 445 939 and in Grinnell et al., 1987, BioTechnolocv 5:1189-1192. Briefly, the plasmid was transfected into 293 cells, then stable transformants were identified and subcultured. The clones which demonstrated X'8833 -8- Do the highest le.el of expression were then grown in a 2 core Opticell fermenter in anchorage-dependent mode,, Serum free .media wa.s Usgid for all fermentations. To prepare the serum 1 free fedia, the f.ollowing ingredients were dissolved in 100 liters of high quality water: Dulbecco. Modified Eagle media (0200-2010) 1350 g .Ham"s F12 media (#63N3085) 478 g Sodium Bicarbonate 432 g S10 Dextrose 468 g Ethanolamine 55 ml Sodium Selenite 0.5M 180 ml 0 Vitamin K 180 ml L-Glutamine 81 g ft i The solution was brought to 180 liters and the pH was ,,,adjusted to 7.2 with 3N HC1 After fermentation at 37 degrees C, the human protein C can be separated from the Sculture fluid by the techniques of Yan, U.S. Patent No.

4,981,952. The human protein C so produced can be used in the unactivated zymogen form or can be activated by procedures well known to one skilled in the art.

Examlie 2 Olicosaccharide Structure of rHPC Produced in 293 Cells Th"'l Many of the procedures used to elucidate the novel structure of the rHPC produced in 293 cells are described in Yan et, al., 1990, BioTechnolov 8: 655-651.

The desalted and lyophilized rHPC was first reduced and carboxymethylated. in 0. 2M Tris buffer (pH8. 6) containing 2mM EDTA and 6M gu.anidine HC1. This solution was thoroughly dialyzed against 50mM ammonium bicarbonate then lyophilized. The denatured rHPC was resuspended in sodium a ayo hepoeuesue oelcdt h ies o~h2~ M9a~~ae X-8833 -9- Sphosphate buffer (pH 8.6) then 12 units of N-glycanase (Genzyme) were added for every 8-9 mg of rHPC. The solution was incubated at 37 degrees C for 72 hours then the enzymatically released N-linked oligosaccharides were separrted from the deglycosylated rHPC on a Bio-gel column equilibrated with 50 mM ammonium bicarbonate.

Fractions containing deglycosylated rHPC were monitored by

OD

280 nm- The fractions containing the oligosaccharides, as monitored by glycosyl composition analysis, were pooled and lyophilized. The oligosaccharides were separated on the Dionex HPAE-PAD II system using a AS6 column (4.6 x S250mm) with an AG6 guard column which was equilibrated with mM sodium acetate, 100 mM NaOH. After 3 minutes, the sodium acetate was increased to 60 mM in 20 minutes, further increased to 200mM in 120 minutes, increased to 400 mM in 40 minutes and again increased to 800mM in 5 minutes.

The NaOH concentration was kept constant at 100mM and the column flow rate was 1 ml/min. Post-column 0.3 M NaOH was flowed at 0.5 ml/min.

An anion micromembrane suppressor was placed S after the PAD detector. 30mM sulfuric acid flowing at 9 ml/min through the anion micromembrane suppressor was sufficient to neutralize and partially desalt the eluant 25 from the AG6-AS6 column. Each oligosaccharide peak was further desalted with an OnGuard H cartridge (Dionex) prepared according to manufacturer's directions.

About 1-10 gg of desalted oligosaccharide was J 0a used as starting material to prepare PMAA derivatives for linkage analysis. The oligosaccharide was pre-reduced with microliters of 10 mg/ml NaBH4 in 1M NH40H for 2 hours at room temperature. The reaction was stopped with microliters of glacial acetic acid, then the borate salts were removed by repeated coevaporation under nitrogen with 10% acetic acid in methanol. The reduced oligosaccharide I- I p f X-8833 was permethylated with a modified procedure of Ciucanu and Kerek, 1984, Carbohvdr. Res. 131:209-217 as described by Costello and Vath, 1990, Meth. Enzvmol. 193:738-755. The permethylated oligosaccharide was hydrolyzed in 2M TFA at 100 degrees C for 2 hours, then dried with rotorevaporation. Deuteroreduction was achieved by adding NaBD4 in 1M NH40H at a concentration of 20 mg/ml at 40 degrees C for 1.5 hours. The reaction was stopped by neutralization with glacial acetic acid. The borate salts were removed by co-evaporation under nitrogen with 10% acetic acid in methanol. Acetylation was carried out by incubation with acetic anhydride at room temperature for 30 minutes. The final PMAA derivatives of the oligosaccharide were extracted with methylene chloride, concentrated by drying under nitrogen and redissolved in 10-20 microliters of ethyl acetate.

Gas Chromatography-Mass Spectrometry (GC-MS) was Scarried out using a Hewlett Packard GC5890-MSD. The PMAA derivatives were dissolved in ethyl acetate prior to injection on a DB-5 column (0.25mm x 30m, J W Scientific) at a helium flow rate of 1 ml/min. The sample was injected at a column temperature of 80 degrees C which was kept for 2 minutes, then increased to 150 degrees at 30 degrees C/minute, then again increased from 150 degrees C to 240 t 25 degrees C at 2 degrees/minute, and kept at 240 degrees C for 10 minutes. The mass spectrometer was run at the El quadruple mode. The GC-MS was calibrated with standards for elution times.

Ai, o The structure of the oligosaccharide of Peak 1 was elucidated by glycosyl composition, linkage analysis and 1H-NMR. The structures of th ther ten major oligosaccharides were predicted on the basis of glycosyl I composition and linkage analysis. The oligosaccharide structures thus elucidated are set forth below.

hA 'bit' The Carbohydrate Structure of Peak No. 1 Fucc(1 GaINAc3(1 -4)GIcNAcj3(1 -*2)Manac(1-*6) Fuf(1 l-*6) Man3(1 -*4)G~cNAcB(1 -*4)GIcNAc GaINAcp3(1 -*4)GcNAc3(1-2)Manx(1 -3) Fucax(l-*3) The Carbohydrate Structure of Peak No. 2 Fuccc(1 GaINAc3(1 -*4)GlcNAcj3\ I-*2)Manac(1-*>6) Fufa(1 Man3(1-*4)GlcNAcp(1 -*4)GlcNAc GaNAc3(1 -*2)Man(1 The Carbohydrate Structure of Peak No. 7 NeuAccL(2-+6)GaINAc(l -*4)GlcNAc(( -*2)Mana(1. Fu]Cc(1-*6) Man3(1 -+4)G~cNAcp3(1 -*4)GlcNAc GaNAcp(1 -*4)GcNAc3(1 -*2)Manx(1 Flimc(1 4~4 C

CIII

It CCItt' C I i The Carbohydrate Structure of Peak 9 CI I I C C CI I C

CCII

:1 1 CCC C.

NeuAcctc(2--*6)GaNAc3(1 -*4)G~cNAcP3(1 *2)Manc(1 Fuc(1-*6) Man3(1 4)GlcNAc3(1 -*4)GlcNAc GaINAcf3(1 -*4)G~cNAcp(1 -*2)Manac(1-*>3) IN:\LIBVV]00439:KEH F- 7~ 12 The Carbohydrate Structure of Peak 11 NeuAcca(2-*3)Galp(1 -*4)GIcNAcp(1 -42)Mantx(1 Fuca( 1 IanI31-*4)G~cNAcp(1 -*4)Gl6NAc GaINAcI3(1 -*4)G~cNAcp(1-+2)Man(1 Fucct(1-*3) The Carbohydrate Structure of Peak NeuAcax(2-*6)GaNAcp(1 -*4)GcNAc3(1 -*2)Mana(1 FucciQ Manp(1 ->4)GI&NAc Ne uAca(2-*3)GaI p(1 -*4)G~cNAcp(1 -*2)Mana(1 The Carbohydrate Structure of Peak 19 NeuAcc(2--*6)Gal3(1 -*4)GcNAc3(1 -*2)Manmx(1-*6) Fuccq(1-->6) Manp3(1 -*)G~cNAcp(1 -*4)G~cNAc NeuA.ctx(2-*3)Gaip(1 -*4)G~cNAc j(1 -*2)Man(1 l-*3) The Carbohydrate Structure of Peak NeuAca(2-*6)GaINAcp(1 -*4)GIcNAc3(1->6) Fuca j1->6) NeuAcax(2-3)Gal3(1 -*4)GIcNAc,(1 Man3(1 -*4)G~cNAc NeuAca(2-3)Gal3(1 -+4)GcNAc3(1 ->2)Manx(1 3) C 4 i t 41 LN:kL]BVV]00439:KEH

I-

'A

13 The Carbohydrate Structure of Peak 23 NeuAcai(2--*6)GaINAc3(1 -*4)G~cNAcp(1 1aFuca1 NeuAca(2-3)Gal3(1 -*4)GlcNAcp3(1-*2)Fuc1-6 Ma[(1 -*4)G~cNAc3(1 -+4)G~cNAc NeuAcca(2--*3)Ga1p(1 -*4)G~cNAcpj(l1 M anca(1- 3) NeuA,,cc(2-3)Gap(1 *4)GIcNAc3(1 2) The Carbohydrate Structure of Peak 24 v 4' NeuAccu(2-6)Gal3(1 -*4)G~cNAcp(1 MancL(1 NeuAcca(2-*3)Gal3(1 -*4)G~cNAcp(1 Iuc(1-6 Manp(1 -+4)G~cNAcp(1 -*4)GIcNAc NeuAccc(2-3)Gapf(1 -*4)G~cNAc j(1-*4) Manx(1 NeuAccc(2-3)GaI3(1 -*4)G~cNAcp(1 44 44 Cs'.

4 4 I INA\LIBVVIO0439:KEH m 14 The Carbohydrate Structure of Peak NeuAca(2-3)Galp(l->4)GlcNAcp(1-->6) Mana(1->6) NeuAca(2->3)Galp(1->4)GlcNAcp(l->2) Fuca(1 -6) Manp(l->4)GlcNAc(1-->4)GlcNAc NeuAca(2->3)Galp(l->4)GlcNAcp(1->4) Mana(1-+3) NeuAca(2->3)Galp(1 ->4)GlcNAcp(1 Example 3 In vitro Cell Adhesion Assays Human Umbilical Vein Endothelium Cells (HUVEC) or Human Aortic Endothelium (HAE) were obtained from Clonetics (San Diego) and grown in the EBM medium supplied by Clonetics. Cells were plated in 96 well plates at a density to obtain confluent monolayers following overnight incubation at 37 degrees C. Monolayers were incubated with or without 20 nanograms Tumor Necrosis Factor (TNF) for 4 to 6 hours prior to the lo binding assay in a total volume of 100 to 150 microliters. To test for inhibition of binding, samples of protein C or its isolated carbohydrates were added to triplicate wells in volumes up to 20 microliters in Phosphate Buffered Saline (PBS) or water, and then incubated for an additional 20 to 25 minutes. Following incubations, tritium-labelled SU937 cells were added in 50 microliter volumes at from 1 to 3 x 10(6) cells per well.

The U937 cells were tritium labelled by the addition of 3H-thymidine to a final concentration of 1 microcurie per milliliter, followed by 18 to 20 hours incubation. Cells were washed with PBS prior to use to remove excess label.

o #e [N:\LIBVVI00439:KEH

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I- ~i~~i X-8833 After a 20 minute incubation of the labeled U937 cells with the endothelial cells, the wells were aspirated and washed four times with calcium-containing PBS. The monolayer and adherent U937 cells were solubilized by the addition of 0.25% SDS/0.1 N NaOH for 5 minutes with agitation. The level of binding was determined by scintillation counting of the solubilized cells.

Table I Experiment# 1(HUVECS)b 1(HUVECS) 2(HAE) 2(HAE) 3(HAE) aPC La/ml 32 120 120 240 120 inhibitiona None 70(12)c None 77(30) 54(18) 4* 1~ *4t a

CC..

a 100 inhibition is the difference between U937 binding in the presence and absence of pretreatment of the endothelial cells with TNF bcell line Cnumbers in parenthesis indicate standard error ir C 'I 1 1 L; -i X-8833 Table I Exp eriment 1 (HAE) b 1 (HAE) 1IH~.' 2 2 (HU-VECS) oliarosaccharide uX 5.75 11.5 23 17.25 34.5 inhibitiona None 22 0 5 )c 16 (4) 19 d 63 a100% ir.hibltion is the difference between U937 binding in the presence and absence of pretreatment of the endothelial cells with TNF bcell line cniumers in parenthesis indicate standard error dno error calculated from single point determinations its I, t tee.

4 I 4* Ii teat

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C

*4 Ct C I

C

4* Ct o i a, p C C, 44 404t40

C

Claims (9)

1. An oflgosaccharide of the structure GaINAc3(1 -+4)GIcNAcI1-+2)Manoc(1->6) FucCL(1 Manp3(I ->4)GIcNAc3(1 -+4)GI&NAc GaNAc3(1 >4)GcNAc3(I ->2)Mana(I1 FuccL(->3).

2. An oligosaccharide of the structure GalNAcf3(1 ->4)GIcNAc3(1->2)Manx(1-*>6) Fufcx(1 6) Manp(1 ->4,)GlcNAcf3(1->4)GI6NAc Ga1NAcpI31->2)Man(1--->3).

3. An oligosaccharide of the structure NeuAcax(2--+6)GaNAcp3(l ->4)GIcNAcp3(1 ->2)Manxc(1->6) Fuca(Q 6) Manf3(1 ->4)GIcNAcp(1--*4)GIcNAc GaJNAcp(I ->4)GcNAc3(1 2)Manc(1

4. An oligosaccharide of the structure NeuAc*(2-->3)Galf3(l 44)GIcNAc j(1 -*2)Manox(1 Fuca(q Man3(1 ->4)GcNAc3(1 4)GI6NAc 'I GaINAc3(1->4)G~cNAcp3(l->2)1Manmx(l-->3) Fuca(1-*>3). 4* O 9 44 4 (NzkLIBVV1O0439:KEH p. II X-8833-(AP) 18 A pharmaceutical formulation comprising as an active ingredient an oligosaccharide as claimed in any one of Claims 1 to 4, associated with one or more pharmaceutically acceptable carriers, excipients or diluents therefor.

6. A method of treating inflammation in a mammal which comprises administering to that mammal an oligosaccharide as claimed in any one of Claims 1 to 4.

7. A method of treating cellular adhesion in a mammal which comprises administering to that mammal an oligosaccharide as claimed in any one of Claims 1 to 4. ooe 0 0 0o I i o r! o fi O Oi? i. I c. >1 19

8. A process of preparing an oligosaccharide of the structure Fucai(1-*>3) GaJNAcf3(1 -*4)GlcNAcI3(l-->2)Manc~l-*6) Fura(l-*6) ManJ3(l -*4)GIcNAc3(1-*4)GIcNAc GalNAcf3(1 -*4)G~cNAcP(1 -*2)Manc(1 Fuca(1 which process is substantially as hereinbefore described with reference to Example 1 or 2.

9. A process of preparing an ohigosaccharide of the structure Fucca(l GaNAc3(1-4)GIcNAcB(l -*2)MancL(l Fucaq( Manf3(l ->4)GlcNAcP(l ->4)GlcNAc GaINAcP3(l-2)Mana(l-*3) which process is substantially as hereinbefore described with reference to Example 1 or 2. It A process of preparing an oligosaccharide of the structure NeuAccx(2-*6)GaNAcp(l--4)GcNAcp3(l lnc(l Fucac(1-*6) Man (1 *)GcNcl -*4)GlcNAc GaINAcp(l *4)GcNAc3(1 -*2)Manx(l -*3)4 Fucc(l-*3) *1 which process is substantially as hereinbefore described with reference to Example 1 or 2.

11. A process of preparing an oligosaccharide of the structure NeuAccx(2-3)Gapf(l -*4)G~cNAcP(l -*2)Manc(l Fuccc( *~,:ManP(l -*4)GlcNAcP(1 -*4)GlcNAc GaINAcI3(1 -*4>)GlcNAcp(1-*2)Manx(l Fuca(1 which process is substantially as hereinbefore described with reference to Example 1 or 2. D)ated 1 June, 1995 Eli Liiiy and Company ~,.<SA~i~,Patent Attorneys for the Applicant/Nominated Person Uj SPRUSON FERGUSON (N-.LIBVV]00439:KEH N, Method For Producing Carbohydrates Abstract The invention relates to molecular biology, particularly to methods for the production of novel carbohydrate structures in human kidney 293 cells. These novel carbohydrate structures can be used to treat inflammation and can also be used to reduce cell adhesion. The oligosaccharides of the invention have the structures: Manf3(i-- 4)GlcNAcJ3(l-- 4)GlcNAc Ga1NAcp3(l 4)G Nc( 2)Mano(1- ft 3 GalNAc3(1 4)GlcNAc3(1 2)Manc(1-. 6) Fuccxl--b6) 4405 1 Man 3(1 .4)GlcNAcJ3(1-b.4)GlcNAc GalNAc3(1 i 3) 00NeuAccx(2 -6)Ga1NAc3(1--.4)G~cNAc3(l1.... 2)Manoc(1--.. 6) Fuccx(1 6) I Manp(-- 4)GlcNAcp(1- 4)GIcNAc Ga1NAcP3(1--4)GcNAc3(1 -2)Man(L.3 FuCc(--3) or 2t NeuAccx(2 3)Galf3(1 4)GlcNAcP(I -t2)Manx(I--. 6) Fucc(1-*'6) Man( I31-4)GlcNAc3(1---b4)GlcNAc Fuca(1-3) ILIbWI\00093:GSA 1 of 1